PROJECT SUMMARY Recent advances in regulatory genomics, especially 3D genome organization in cell nucleus, suggest that existing methods for cross-species comparisons are limited in their ability to fully understand the evolution of non-coding genome function. In particular, it is known that genomes are compartmentalized to distinct compartments in the nucleus such as nuclear lamina and nuclear speckles. Such nuclear compartmentalization is an essential feature of higher-order genome organization and is linked to various important genome functions such as DNA replication timing and transcription. Unfortunately, to date no study exists that directly compares nuclear compartmentalization between human and other mammals. In addition, there are no computational models available that consider the continuous nature of multiple features of nuclear compartmentalization and function, which is critical to integrate genome-wide functional genomic data and datasets that measure cytological distance to multiple compartments across species. In this project, we will develop novel algorithms and generate new datasets to directly address two key questions: (1) How to identify the evolutionary patterns of nuclear compartmentalization? (2) What types of sequence evolution may drive spatial localization changes across species? The proposed project represents the first endeavor in comparative genomics for nuclear compartmentalization. Our Specific Aims are: (1) Developing new probabilistic models for identifying evolutionary patterns of nuclear compartmentalization. (2) Identifying genome-wide evolutionary patterns of nuclear compartmentalization in primate species based on TSA-seq and Repli-seq. (3) Developing new algorithms to connect sequence features to nuclear compartmentalization through cross-species comparisons. Successful completion of these aims will result in novel computational tools and new datasets that will be highly valuable for the comparative genomics community. Integrating the new computational tools and unique datasets will provide invaluable insights into the relationship between sequence evolution and changes in nuclear genome organization in mammalian species. Therefore, the proposed research is expected to advance comparative genomics to a new frontier and provide new perspectives for studying human genome function